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1.
2.
A mixture of differentially labeled mouse and Bacillus subtilis DNA was used as a source of oligodeoxynucleotides of chain lengths from 15 to 40 nucleotides. The extent of interaction of these oligonucleotides with homologous or heterologous DNA bound to membrane filters was measured. The specificity of such interactions increases with chain length and with the incubation temperature. The thermal stability of the complexes is a function of chain length. Homologous oligonucleotide/DNA duplexes of B. subtilis are more stable than those of mouse of corresponding size, consistent with the incidence of partially related base sequences in mouse DNA. Oligonucleotides in this size range are also able to recognize partially complementary base sequences in the DNA of closely related organisms. This approach shows promise as a means of obtaining quantitative estimates of base sequence divergence between the DNAs of related organisms.This research was supported by a grant from the National Science Foundation (GB 6099).  相似文献   

3.
The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.  相似文献   

4.
An in-depth study into the incorporation of multiple 3′-S-phosphorothiolate modifications into oligodeoxynucleotides (ODNs) and their subsequent effect on ODN/DNA and ODN/RNA duplex stability. 3′-S-Phosphorothiolate linkages increase the stability of ODN/RNA duplexes and decrease the stability of ODN/DNA duplexes.  相似文献   

5.
An in-depth study into the incorporation of multiple 3-S-phosphorothiolate modifications into oligodeoxynucleotides (ODNs) and their subsequent effect on ODN/DNA and ODN/RNA duplex stability. 3-S-Phosphorothiolate linkages increase the stability of ODN/RNA duplexes and decrease the stability of ODN/DNA duplexes.  相似文献   

6.
The thymidine monomers of LNA with both alpha- and beta-configuration are incorporated with polarity reversals (i.e., with 3'-3' and 5'-5' junctions) in oligodeoxynucleotides with beta- and alpha-configuration, respectively. A 5'-O-phosphoramidite of the beta-LNA monomer is synthesised. Large destabilisations of duplexes with both complementary DNA and RNA are observed for oligodeoxynucleotides containing the alpha-LNA monomer, whereas a duplex with complementary RNA of an alpha-oligodeoxynucleotide containing the beta-LNA monomer is not destabilised.  相似文献   

7.
Wang Z  Shi J  Jin H  Zhang L  Lu J  Zhang L 《Bioconjugate chemistry》2005,16(5):1081-1087
Antisense oligonucleotides are recognized to be very efficient tools for the inhibition of gene expression in a sequence specific way. For the discovery of a novel efficient way to modify oligonucleotides, a series of single isonucleotide-incorporated antisense oligodeoxynucleotides have been synthesized, in which an isonucleotide was introduced at different positions of the sequences. The binding behaviors of modified oligodeoxynucleotides to the complementary sequence were studied by UV, CD, and molecular dynamics simulation. The results showed that although the incorporated isonucleotides at certain positions of the sequence interfere with the binding ability to a different extent, B-form duplexes were maintained and the binding abilities of the 3'-end-modified duplexes were better than the corresponding mismatched duplexes. The digestion of modified oligodeoxynucleotides by snake venom phosphodiesterase showed that an isonucleotide strongly antagonizes hydrolysis. The DNA/RNA hybrid formed by a modified oligodeoxynucleotide and its target RNA could activate RNase H. The 3'-end-modified antisense oligodeoxynucelotides inhibited S-glycoprotein expression of SARS-CoV at the mRNA levels in insect Sf9 cells. This study indicated the possibility of designing a novel and effective antisense oligodeoxynucleotide by incorporating an isonucleotide at the 3'-end of the sequence.  相似文献   

8.
5-Carboxy-2'-deoxycytidine (dC(COO-)) was synthesized as an anion-carrier to seek a new possibility of modified oligodeoxynucleotides capable of stabilization of duplexes and triplexes. The base pairing properties of this compound were evaluated by use of ab initio calculations. These calculations suggest that the Hoogsteen-type base pair of dC(COO-)-G is less stable than that of the canonical C+-G pair and the Watson-Crick-type base pair of dC(COO-)-G is slightly more stable than the natural G-C base pair. The modified cytosine base showed a basicity similar to that of cytosine (pKa 4.2). It turned out that oligodeoxynucleotides 13mer and 14mer incorporating dC(COO-) could form duplexes with the complementary DNA oligomer, which were more stable than the unmodified duplex. In contrast, it formed a relatively unstable triplex with the target ds DNA.  相似文献   

9.
DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes.  相似文献   

10.
C Goffin  V Bailly    W G Verly 《Nucleic acids research》1987,15(21):8755-8771
Using synthetic oligodeoxynucleotides with 3'-OH ends and 32P-labelled 5'-phosphate ends and the technique of polyacrylamide gel electrophoresis, it is shown that, in the presence of the complementary polynucleotide, an AP (apurinic or apyrimidinic) site at the 3' or the 5' end of the labelled oligodeoxynucleotides does not prevent their ligation by T4 DNA ligase, although the reaction rate is decreased. This decrease is more severe when the AP site is at the 3' end; the activated intermediates accumulate showing that it is the efficiency of the adenyl-5'-phosphate attack by the 3'-OH of the base-free deoxyribose which is mostly perturbed. Using the same technique, it is shown that a mispaired base at the 3' or 5' end of oligodeoxynucleotides does not prevent their ligation. A one-nucleotide gap, limited by 3'-OH and 5'-phosphate, can also be closed by T4 DNA ligase although with difficulty; here again the activation of the 5'-phosphate end does not seem to be slowed down, but rather the 3'-OH attack of the adenyl-5'-phosphate. All these anomalous ligations take place with the nick or the gap in front of a continuous complementary strand. Blunt ends ligation of correct duplexes occurs readily; however an AP site or a mispaired base at the 3' or 5' end of one strand of the duplexes prevents ligation between these strands. But a missing nucleotide (responsible for one unpaired nucleotide protruding at the 3' or 5' end of the complementary strand) does not stop ligation of the shorter oligodeoxynucleotides between independent duplexes.  相似文献   

11.
Synthetic oligodeoxynucleotides are widely used in many biological, biochemical and biophysical applications. The concentration, composition and structure of DNA are often determined from its ultraviolet spectrum. Although parameters for use with the nearest-neighbor model for prediction of extinction coefficients of single stranded DNAs at 260 nm were published some time ago, similar parameters for other wavelengths or for use with DNA duplexes have not been reported. Practical formulae and parameters for prediction of UV spectra, hypochromism and peak wavelengths were experimentally determined for both single stranded and double stranded oligodeoxynucleotides in the range from 215 to 310 nm. The accuracy of predictions made using the nearest-neighbor model and the base composition model was determined and compared. The spectrum of any DNA oligomer can be calculated using a Microsoft Excel application that is available in the Appendix A.  相似文献   

12.
2-(1-Naphthalenylethynyl)-2'-deoxyadenosine ((N)A) was synthesized and incorporated into oligodeoxynucleotides. DNA duplexes containing newly designed 5'-(N)AT-3'/3'-T(N)A-5' base pairs are considerably stabilized than unmodified duplexes by stacking interaction of naphthalene rings in the narrow minor groove as characterized by a new emission at longer wavelength and exciton coupled CD signals.  相似文献   

13.
A new synthesis route for long phosphate-methylated oligodeoxynucleotides is described, which were used as antisense inhibitors of the DNA replication. Phosphate-methylated oligomers hybridize more strongly with natural DNA than their natural analogues, due to the absence of electrostatic interstrand repulsions. Compared with phosphate-ethylated and methyl phosphonate systems, phosphate-methylated systems are preferable as antisense DNA, which was concluded from the high Tm values and sharp melting transitions of duplexes of phosphate-methylated and natural DNA. By using the Sanger dideoxy technique, it was shown that a complementary phosphate-methylated 18-mer can effectively and site-specifically block the DNA replication process at room temperature.  相似文献   

14.
To construct the nuclease-resistant oligodeoxynucleotides (ODNs) with natural phosphodiester linkages, we synthesized ODNs that contain 6'alpha-[N-(aminoalkyl)carbamoyloxy]-carbocyclic-thymidines (4, 5, and 6). The stability of these ODNs to nuclease hydrolysis was examined by using snake venom phosphodiesterase (3'-exonuclease) and nuclease S1 (endonuclease). It was found that the ODNs containing 4, 5, or 6 were more resistant to both the enzymes than the unmodified ODN. These nuclease-resistant properties are noteworthy, since they have natural phosphodiester linkages. Next, the thermal stabilities of duplexes consisting of these ODNs and either the complementary DNA or RNA were studied by thermal denaturation. The ODNs that contain 4 were found to enhance the thermal stability of the duplexes with the complementary DNA, while those containing 5 or 6 decreased the thermal stability of the ODN-DNA duplexes. On the other hand, all ODNs that contained 4, 5, or 6 decreased the thermal stability of the ODN-RNA duplexes.  相似文献   

15.
Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.  相似文献   

16.
The immobilization of thiol-derivatized DNA on a Au (111) single crystal surface by self-assembly has been investigated by electrochemical scanning tunneling microscopy (EC-STM). Continuous potential-dependent orientation changes of double-stranded oligodeoxynucleotides (ODN) have been observed in a certain potential range from 200 to 600 mV (versus SCE). It is suggested that the DNA duplexes stand straight on the gold surface at potentials negative of the potential of zero charge (pzc) and then lay down on the surface when the potential shifts positively. These results are in agreement with the expectation based on the Coulombic interaction consideration between negatively charged DNA helices and gold surface. As the applied potential shifts positively, the surface charge changes from negative to positive, that is, the Coulombic force between negatively charged DNA helices and gold surfaces changes from repulsion to attraction. However, for the single-stranded oligodeoxynucleotides, no distinct changes in the surface structure were observed with the applied potential.  相似文献   

17.
7-Deaza-2'-deoxyadenosine and -guanosine phosphoramidite building blocks as well as corresponding 5'-triphosphate derivatives are described carrying in position 7 substituents such as iodo, hexyn-1-yl or 5-aminopentyn-1-yl residues. The phosphoramidites were used to synthesize a series of modified oligodeoxynucleotides. A systematic study of the thermal stabilities of these oligonucleotide duplexes demonstrated that the 7-substituents are well accommodated in the major groove of B-DNA. The 7-(aminoalkyn-1-yl)-7-deazapurine 2'-deoxynucleoside triphosphates were labeled with bulky fluorophores such as Rhodamine Green(R) or tetramethylrhodamine.  相似文献   

18.
2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA.  相似文献   

19.
Phenylalkyl modified phosphoramidites (alkyl chain length n = 1, 2, 3, 5; Fig. 1) were synthesised and incorporated into a DNA hexamer (5'-d(GCCp-GCG); p = place of modification). The obtained diastereomeres were separated by RP-HPLC. After hybridisation with the complementary DNA strand Tm-value and thermodynamic data were measured. The stability of duplexes depends on the linker length and the absolute configuration of the backbone modified oligodeoxynucleotides (Rp, Sp).  相似文献   

20.
John DM  Weeks KM 《Biochemistry》2002,41(21):6866-6874
2'-Amine-substituted nucleotides in hybridized duplexes can be chemically tagged in an acylation reaction that is faster for mismatched or flexible nucleotides than for residues constrained by base pairing. Here we explore mismatch and hybridization detection using probe oligodeoxynucleotides containing single 2'-aminocytidine or -uridine nucleotides annealed to DNA or RNA targets under nonstringent conditions, below T(m). Consistent with a mechanism in which 2'-amine acylation is gated by local nucleotide flexibility, we find that efficient acylation is correlated with formation of weaker or fewer hydrogen bonds in base pair mismatches. Using 2'-aminocytidine-containing probes annealed to both DNA and RNA targets, mismatches are reliably detected as rapid selective acylation of the 2'-amine group in two sequence contexts. For probe oligonucleotides containing 2'-aminouridine residues, good discrimination between U-A base pairs and U-G mismatches could be obtained for DNA-DNA but not for DNA-RNA duplexes upon the introduction of a single 2'-O-Me group 5' to the 2'-amino nucleotide. The 2'-O-Me group introduces a structural perturbation, presumably to a more A-form-like structure, that exaggerates local flexibility at mismatches in DNA strands. Thus, 2'-amine acylation can be used to interrogate all possible mismatches in DNA-DNA duplexes and mismatches involving 2'-amine-substituted cytidine nucleotides in DNA-RNA heteroduplexes. Applications of this chemistry include detecting and chemically proofreading single nucleotide polymorphisms in both DNA and RNA targets and quantifying absolute amounts of RNA.  相似文献   

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